This invention relates to a process for the production of compound (+) catechin penta acetate useful as a precursor for the production of (+) catechin. More particularly, this invention relates to a process for the production of (+) catechin penta acetate of formula (1) from Taxus wallichiana tissue cultures which is useful as a precursor for the production of (+) catechin of formula (2). 
(+) catechin peta acetate of formula (1) can be prepared only from (+) catechin of formula (2) through acetylation. Therefore, the only source to get (+) catechin penta acetate consists of two processesxe2x80x94first to isolate (+) catechin and then to convert it into (+) catechin penta acetate through acetylation. However, in this invention, the precursor (+) catechin penta acetate can be directly obtained from the cell cultures of Taxus wallichiana following the process of the process of the present invention. Moreover, (+) catechin of formula (2) is a polyphenolic compound and is susceptible to aerial oxidation and forms mixture of compounds on exposure to air. However, (+) catechin penta acetate of formula (1) is a stable molecule and can withstand aerial oxidation. Thus, it has a high self life and can be useful as a precursor for the production of (+) catechin.
In recent years, different Taxus species have attracted world wide attention due to the presence of taxol or its analogues in the bark or needles of the trees. Taxol, a highly oxygenated diterpenoid molecule and a potent anticancer drug was first isolated from the stem bark of Texus brevifolia. Thereafter, it ahas also been isolated from other Taxus species including T. wallichiana. 
Catechins, the basic structural unit of condensed tannins, belong to flavan-3-ol derivatives and are found in a wide variety of plant sources such as vegetables, herbs and teas (Phytochem (1981) 20:869). Considerable interest have been expressed regarding the various pharmacological functions of catechins, which have been proved to be antibacterial, antiviral, antitumour, antioxidant and radical scavengers (Phytochem (1998) 49:2379-82).
The direct manipulation of plant cell and tissue culture systems has resulted in an enhanced production of various secondary metabolites. In vitro production of catechins, mainly (xe2x88x92) epicatechin-3-O-gallate accompanied by (+) catechin and (xe2x88x92) epicatechin have been reported in Fagophyrum esculentum calli and hairy root cultures (Phytochem (1992) 3 1:1239-1241; Phytochem (1993) 32:929), suspension cultures of Camellia sinensis (Chayekexue (1995) 15:111-116) and Vitis vinifera (Biotech Lett. (1996) 659-662). Crataegus monogyna (Phytochem (1994) 37:1273), Uncaria elliptica (Phytochem (1998) 28:1099-1100) and Polygonum hydropiper (Phytochem (1998) 49:1935-39).
In a prior art process, (+) catechin was isolated from the cell cultures of T. wallichiana with a yield of 0.3%, which comprises inoculation of explants on different media compositions supplemented with combinations of auxins (1-5 mg/l) and cytokinins (0.1-1.0 mg/l), incubation of the cultures under continuous light or dark conditions for 4-6 weeks for callus initiation followed by subculturing at 4-6 weeks intervals, extraction of fresh pulverized calli with polar solvents at room temperature, evaporating the polar solvent to give a residue, and treatment of the residue with a chlorinated solvent and isolation of (+) catechin by filtration (SK Chattopadhyay et al. Indian Patent Application No. 215/DEL/2000, applicants Ref. No. NF364/99).
Several approaches have been used for the establishment of in vitro cultures of various Taxus species (Plant Cell, Tissue and Organ Cult. (1996) 46:59-75). Different explants and various basal media have been used for initiation and maintenance of Taxus callus and cell suspension cultures. The culture media are frequently supplemented with organic substances, such as casein hydrolysate, polyvinylpyrrolidone, ascorbic acid and others. Several growth regulators are used for stimulation of cell proliferation.
Taxus wallichiana, known as Himalayan yew is available in India. Suspension and callus cultures of T. wallichiana are found capable of producing taxol (Planta Med. (1998) 64:270-72) and some important taxanes, namely 2-deacetoxy-taxinine J and 2-deacetoxy autrospicatin (Planta Med. (1996) 62:333-35). We have been screening different callus lines of T. wallichiana induced from different explants of tress collected from different geographical regions of India. The protocol standardized for in vitro callus production is dependent on media composition (viz. Murashinge and Skoog""s Gamborg""s, White""s, Nitsch and Nitsch""s), hormonal regime combinations of different concentrations of cytokinins and auxins, (such as 6-benzyl aminopurine, TDZ, 2-ip, Kinetin, 6-methylamino purine, Zeatin with NAA, IAA, IBA, 2,4-D, 2,4-T, Picloram), explant source (preferably from needles, twigs, stems devoid of needles and seeds) and culture conditions (light dark conditions).
The callus line developed from a specific explants on different media compositions having definite hormonal combinations resulted in the production of a compound. The compound, having a molecular formula C25H24O11, mp 131-132xc2x0 C., was isolated as a crystalline solid with a yield of 0.05%. The compound has been characterized as (+) catechin penta acetate.
The (+) catechin penta acetate is a very important precursor for the synthesis of catechin which in turn can be converted into its other biologically active derivatives, eg. gallocatechin, epigallocatechin and epigallocatechin-3-o-gallate.
There has not been any process reported in the literature for direct isolation of (+) catechin penta acetate either from higher plants, microbes or from cell cultures of plants.
Thus, the drawbacks of the previous processes for the preparation of (+) catechin penta acetate are as under:
These processes require isolation of (+) catechin and the convertion thereof to catechin penta acetate through acetylation process.
(+) catechin can not be preserved for a longer period of time due to its instability towards aerial oxidation. However, (+) catechin penta acetate can be preserved without its decomposition for a longer period of time and it easily can be converted into (+) catechin with a quantitative yield.
Thus the main object of the present invention is to provide a process for in vitro production of a compound (+) catechin penta acetate of formula 1 from T. wallichina tissue cultures.
Another object is to provide a process for isolation of (+) catechin penta acetate from cell cultures of T. wallichiana. 
Yet another object is to provide a process for the production of a stable precursor (+) catechin penta acetate from T. wallichiana tissue cultures for the production of (+) catechin.
Still another object is to provide a process for the production of (+) catechin penta acetate from the cell cultures of T. wallichiana which can then be converted into (+) catechin quantitatively.
The present invention consisting of the in vitro production of (+) catechin penta acetate from T. wallichiana constitutes the first ever report of production of (+) catechin penta acetate in the genus Taxus.
In contrast to prior art processes for the production of the precursor (+) catechin penta acetate, which comprises, first isolation of the (+) catechin (which is susceptible to aerial oxidation and polymerization) and then to convert it into catechin penta acetate, this invention provides a process for the direct production of a stable precursor (+) catechin penta acetate in the cell cultures of T. wallichiana. Also, this stable precursor (+) catechin penta acetate can be converted into (+) catechin quantitatively.
Thus, the novelty of the present invention is that it provides a process for the in vivo production of a stable precursor (+) catechin penta acetate in the cell cultures of T. wallichiana, which can be converted into (+) catechin quantitatively.
Accordingly the present invention relates to a process for the production of a compound (+) catechin penta acetate of formula I from T. wallichiana tissue cultures which comprises:
(a) inoculation of explants on different media compositions supplemented with combinations of auxins (1-5 mg/l) and cytokinins (0.1-1.0 mg/l);
(b) incubation of the cultures under continuous light or dark conditions for 4-6 weeks for callus initiation followed by subculturing at 4-6 weeks intervals;
(c) harvesting the calli at different growth phases ranging from 12 to 36 months;
(d) extraction of fresh pulverized calli with polar solvents at room temperature;
(e) evaporating the polar solvent to give a residue; and partitioning of the residue between water and chlorinated solvent and evaporating the solvent to semi-solid mass;
(f) subjecting the resultant mass to column chromatography over suitable adsorbents and (g) eluting with organic solvent/mixtures of organic solvent to get catechin penta acetate.
In an embodiment of the present invention the explants for induction of callus may be selected from needles, twigs, stem devoid of needles and seeds.
In another embodiment of the present invention the culture media for callus induction and multiplication may be selected from Murashige and Skoog; (1962) (MS) medium, containing the following (in mg/l)- NH4NO3 (1,650), KNO3 (1,900), CaCl2.2H2O (400), MgSO4.7H2O (370), KH2PO4 (170), Na2EDTA.2H2O (7.2), FeSO4.7H2O (27.8), MnSO4.4H2O (22.3),ZnSO4.7H2O (8.6), H3BO3 (6.2), KI (0.83), Na2MoO4.2H2O (0.25), CuSO4.5H2O (0.025), CoCl2.6H2O(0.025), Glycine (2.0), Nicotinic acid (0.5), Pyridoxine HCL (0.5), Thiamine HCl (0.1); Gamborg""s (1968) (B5) medium, containing the following (in mg/l)- KNO3 (3,000), (NH4)2SO4 (134), MgSO4.7H2O (500), CaCl2.2H2O (150), NaH2PO4.H2O (150), MnSO4.H2O (10.0), KI (0.75), H3BO3 (3.0), ZnSO4.7H2O (2.0), CuSO4 (0.025), NaMoO4.2H2O (0.25), CoCl2.6H2O (0.25), Na2EDTA.2H2O (37.2), FeSO4.7H2O (27.8); White""s (1963) medium, consisting of the following (in mg/l)- Ca(NO3)2 (142.0), KNO3 (81.0), MgSO4.7H2O (70.0), KCl (65.0), KH2PO4 (12.0), Fe(SO4)3 (2.46) and Nitsch and Nitsch; (1969) medium, containing the following (in mg/l)- NH4NO3 (20.0), KNO3 (950), H3BO3 (10.0), KH2PO4 (68.0), Na2MoO4.2H2O (0.143), CaCl2 (41.5), MgSO4.7H2O (185), MnSO4.4H2O (15.0), ZnSO4.H2O (10.0), CuSO4 (0.14), FeSO4 (111.4), Na2EDTA (149), Biotin (0.05), Glycine (2.0), Nicotinic acid (5.0), Pyridoxine HCl (0.5), Thiamine HCl (0.5), Folic acid (5.0).
In yet another embodiment, the auxins may be selected from indole acetic acid (IAA), napthelene acetic acid (NAA), indole butyric acid (IBA), 2,4-dichlorophenoxy acetic acid (2,4-D), 2,4,6-trichlorophenoxy acetic acid (2,4-T) and picloram within the following range (0.2-20 mg/l).
In still another embodiment, the cytokinins may be selected from 6-benzyl amino purine (BAP), 6-methyl aminopurine (MAP), kinetin (Kn), zeatin, thiadiazuron (TDZ) and 2-isopentenyl amino purine (2-ip) within the following range (0.02-2 mg/l).
In still another embodiment, the cultures may be incubated under continuous light of 300xe2x80x94300 lux or under continuous dark conditions.
In still another embodiment, harvesting time get maximum product may be from twenty four to thirty six months.
In still another embodiment of the present invention, the polar solvents may be selected from methanol, ethanol, propanol and butanol.
In still another embodiment, the ratio of auxin and cytokinin used ranges between 5 to 20:1.
In still another embodiment, the medium may be supplemented with casein hydrolysate ranging between 100 to 400 mg/l.
In still another embodiment, ascorbic acid used ranges between 10-50 mg/l.
In still another embodiment, the chlorinated solvents used for partitioning may be selected from chloroform, dichloromethane.
In still another embodiment, the adsorbents used for column chromatography for isolating catechin penta acetate may be selected from silica gel, alumina and florosil.
In still another embodiment, the organic solvent, mixtures of organic solvents used for eluting the column may be selected from ethyl acetate, chloroform, dichloromethane, hexane-ethyl acetate mixtures, pet.ether-ethyl acetate mixtures, hexane-chloroform mixtures, pet. ether-chloroform mixtures.
Repeated experimentations have proved that use of particular explants and specific ratio of auxins and cytokinins are the critical factors for in vitro expression of (+) catechin penta acetate production.